Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early Drosophila embryo

Positional information in developing tissues often takes the form of stripes of gene expression that mark the boundaries of a particular cell type or morphogenetic process. How stripes form is still in many cases poorly understood. Here we use optogenetics and live-cell biosensors to investigate one such pattern: the posterior stripe of brachyenteron (byn) expression in the early Drosophila embryo. This byn stripe depends on interpretation of an upstream signal – a gradient of ERK kinase activity – and the expression of two target genes tailless (tll) and huckebein (hkb) that exert antagonistic control over byn. We find that high or low doses of ERK signaling produce either transient or sustained byn expression, respectively. These ERK stimuli also regulate tll and hkb expression with distinct dynamics: tll transcription is rapidly induced under both low and high stimuli, whereas hkb transcription converts graded ERK inputs into an output switch with a variable time delay. Antagonistic regulatory paths acting on different timescales are hallmarks of an incoherent feedforward loop architecture, which is sufficient to explain transient or sustained byn dynamics and adds temporal complexity to the steady-state model of byn stripe formation. We further show that an all-or-none stimulus can be ‘blurred’ through intracellular diffusion to non-locally produce a stripe of byn gene expression. Overall, our study provides a blueprint for using optogenetic inputs to dissect developmental signal interpretation in space and time.


Dynamics of an incoherent feedforward loop drive ERK-dependent pattern formation in the early
: The ventral region used in this study does not express endogenous tll, hkb, or byn (A) Plot of total number of nuclei that burst in response to high, low, and no light for tll, hkb, and byn. There is no transcription of any of the targets under no light in this ventral region, indicating that all bursts we observe are induced by OptoSOS. Mean ± SEM, n = 3-7 embryos. (B) Image of the posterior pole of an embryo in the dark expressing Tll LlamaTag (see Figure S3). The viewing region used for all experiments is marked in magenta and is clearly distant from the region of endogenous Tll.

Figure S2: Characterizing byn, tll and hkb transcriptional response to high and low light
We noticed that there was some variability in the expression domains of byn, tll, and hkb between embryos, with the most notable variation seen for the anterior extent of byn and tll expression under low light. (A) To control for this heterogeneity, we quantified only a 50 x 45 µm box at the most anterior portion of expression. byn dynamics in this region were similar between replicates. (B) Plots show variability in byn (orange), tll (teal), and hkb (magenta) expression between embryos. Each plot represents the viewing region (magenta box) of one embryo and each point represents a nucleus that is in the bursting state at the specified timepoint. The chosen quantification region is shown for a few representative embryos (black box). (C-D) Proportion of nuclei in the tll (C) and hkb (D) bursting state through NC14. For the hkb low light condition, only embryos with bursts present were included. Mean ± SEM, n = 4-5 embryos in each condition. Figure S3: Accumulation of Hkb protein is delayed relative to Tll protein (A) CRISPR/Cas9-tagged tll and hkb alleles with an anti-mCherry nanobody (LlamaTag) at the N-terminus. Upon translation of Tll or Hkb protein, the nanobody (dark blue) binds pre-folded mCherry protein (red) in the embryo, leading to accumulation of mCherry-bound Tll/Hkb protein in the nucleus. We generated embryos that expressed the OptoSOS system and mCherry (OptoSOS/mCherry embryos) and which also contained either the Tll-LlamaTag or Hkb-LlamaTag. (B) Schematic showing that illumination with high light begins in NC10 and continues until either 10 min into NC14 ("early") or 25 min into NC14 ("late"). The star shows the point 15 min into NC14 where transient byn transcription has ended. (C) Images show Tll or Hkb LlamaTag at early and late NC14. Nuclear localization indicates the protein is present. Scale bar is 10 µm. (D) Number of LlamaTag+ nuclei in each condition. Each dot represents one embryo. There are significantly more Tll+ nuclei in early NC14 but not in late NC14 by two-way ANOVA and Sidak's post hoc test. Mean ± SEM, n = 4 embryos.

Figure S4: Characterizing expression in the unilluminated region
When comparing the illuminated and unilluminated regions, we noticed that there was variability in the position where the byn stripe forms relative to the illumination boundary. (A) To control for this heterogeneity, we quantified only a 30 x 70 µm box positioned at the furthest extent of byn expression. In the illumination region, the quantification region was fixed 5 µm away from the illumination boundary. (B) Plots show the variability in byn (orange) expression between embryos 15 minutes into NC14. At this timepoint, only nuclei expressing sustained byn are bursting. Each plot represents the viewing region (magenta box) of one embryo and each point represents a nucleus that is in the bursting state. The quantification region is shown for one representative embryo (black box). (C) We also measured expression of byn in the unilluminated region under low light. There was very little expression in the unilluminated region, consistent with ERK levels too low to induce expression. (D) Upon low light illumination, byn-MS2 bursts in NC14 were sustained in the illuminated region and there were very few bursts in the unilluminated region. The blue bar shows the illuminated portion of the viewing window, and the dashed blue line shows the illumination boundary. Orange nuclear masks show nuclei with a burst in corresponding frames. Scale bar is 10 µm. (E) Quantification shows the proportion of nuclei in the byn bursting state through NC14 upon low light stimulation. Mean ± SEM, n = 4 embryos in each condition. Significance from two-way ANOVA with Sidak's post hoc test.

Figure S5: Local Ras activation produces a diffusive gradient of ERK activation and gene expression
We quantified the response of multiple effectors to our high light input on the ventral side. Light was applied from NC10 to NC14 and all measurements were taken in NC14. For each effector a representative image is shown on the left and quantification is shown on the right. Under high light, the SSPB-tagRFP-SOS cat component of OptoSOS is recruited to the membrane only in the illuminated region, with a sharp drop-off at the illumination boundary. Graph shows mean ± SEM intensity at each position from 3 embryos under high light. (C) We used miniCic, a reporter of ERK kinase activity, to assess ERK activity (Moreno et al. 2019). When ERK is off, miniCic is nuclear, and when ERK is on, miniCic relocalizes to the cytoplasm. Under high light, miniCic is cytoplasmic in both the illuminated and unilluminated regions, showing that ERK activity extends into the unilluminated region (upper image). The miniCic reporter saturates at relatively low levels of ERK, and so to understand the shape of the ERK gradient, we also applied low light. Under low light, there is a graded decrease in ERK activity at increasing distances from the illumination boundary (lower image, graph). Thus, a sharp boundary of OptoSOS activity is blurred into a gradient of ERK activity. Graph shows (1 -nuclear miniCic intensity) for n=3 embryos, each dot is one nucleus. (D) In late NC14, nuclear Tll LlamaTag (top) forms a shallow gradient, indicating that Tll protein is present up to 50 µm away from the illumination boundary. Nuclear Hkb LlamaTag (bottom) forms a steeper gradient. Graphs show nuclear LlamaTag intensity for n=3 embryos, each dot is one nucleus. The lines show the quadratic (Tll) and logistic (Hkb) function that best fits the data. (E) Images show tdEOS-tubulin at 0, 5, and 10 minutes post conversion from green to red in an early NC14 (top) and cellularized (bottom) embryo. The photoconverted region is to the left of the dotted white line and the dashed magenta box shows the quantified region 25 to 50 µm away from the illumination boundary. (F) Fold change in tdEOS-Red in the region 25 to 50 µm away from the illumination boundary in NC14 and cellularized embryos. n = 3 embryos for each condition. Mean ± SEM. Keenan et al (2020) were analyzed at the tll and hkb enhancers after OptoSOS stimulation for 0, 5, or 30 min. After 5 min, most Cic has been lost from the tll enhancer, but a peak remains at the hkb enhancer that is not lost until 30 min post stimulation.

LlamaTag crosses and imaging
Female 67/vasa-mCherry; 15/ OptoSOS virgins were mated with LlamaTag males. Embryos were stimulated with high light starting in NC10 on the right half of the ventral side. Under high light, bright membrane recruitment of SSPB-tagRFP-SOS cat obscured nuclear accumulation of the LlamaTags, and so we turned off the blue light for 10 minutes prior to imaging of LlamaTags. For the "early NC14" timepoint, the blue light was turned off when nuclei entered the 13 th mitosis and the LlamaTag was imaged 10 minutes later. For the "late NC14" timepoint, the blue light was turned off 15 minutes into NC14 and the LlamaTag was imaged 10 minutes later. For visualization of the gradient (Figure S5D), the late NC14 timepoint was used.

miniCic crosses and imaging
Embryos from 67/miniCIC-NeonGreen; 15/ OptoSOS mothers were stimulated with high or low light starting in NC10 on the right half of the ventral side. Images were collected in early NC14.

Photoconversion crosses and imaging
Embryos from 67/UASp-alphaTub84B.tdEOS ; 15/ + mothers were treated with bleach for 30 seconds to remove the eggshell, washed thoroughly with water, and mounted for imaging.
Embryos at the start of NC14 or mid-gastrulation (roughly 4 hrs post egg lay) were selected. At one pole of the embryo, the entire field of view was stimulated for 1 minute with a 405 laser at 100% power. Immediately following the conversion, the stage was shifted to reveal the conversion boundary and roughly 5 x 1 μm z stacks were collected at 5-minute intervals.

Quantification of LlamaTags and miniCIC
All images containing LlamaTags were maximum projected with z slices containing the nuclei. Then, the background was subtracted using rolling ball subtraction with radius 50 pixels.
For each embryo, the number of nuclei detectable by nuclear localization of the LlamaTag within the equal-sized viewing region was counted. To quantify the gradient of nuclear LlamaTag, a gaussian blur with radius 2 was applied to the background subtracted images. The intensity at roughly 100 points spanning the illuminated and unilluminated region was measured. These points were selected within nuclei if nuclei were detectable nearby. If there were no detectable nuclei, points were placed randomly. Then, all points within each embryo were normalized from 0 to 1. The gradient of nuclear miniCic was quantified similarly from a single z slice.

Quantification of photoconversion experiments
Images were maximum projected over 2-3 z slices that contained the nucleus and then the background was subtracted using rolling ball subtraction with radius 50. A quantification box was drawn spanning the region 25-50 μm away from the illumination boundary. The level of red tdEOS fluorescence in that box was measured at each timepoint. Then the fold change in red fluorescence compared to the timepoint immediately following the illumination (0 minutes) was determined.

Legends for Supplemental Movies
Movie S1: NC14 byn-MS2 dynamics under high and low light Movie shows byn-MS2 bursts during NC14 under high (top) and low (bottom) blue light. On the left are the raw images and on the right are segmented images. Orange nuclear masks show nuclei with a burst in corresponding frames. Timer (mm:ss) shows time since the start of NC14. Scale bar is 10 µm.

Movie S2: tll-and hkb-MS2 dynamics under high and low light
Sequential movies show tll-MS2 under high light, tll-MS2 under low light, hkb-MS2 under high light, and hkb-MS2 under low light. Each frame is labeled with the corresponding nuclear cycle (NC10-14). Scale bar is 10 µm. Timer (hh:mm:ss) shows time since the blue light was applied.

Movie S3: NC14 byn-MS2 dynamics under high, short light
Movie shows byn-MS2 bursts during NC14 when high light is applied only from NC10-NC13. Orange nuclear masks show nuclei with a burst in corresponding frames. Timer (mm:ss) shows time since the start of NC14. Scale bar is 10 µm.

Movie S4: NC14 tll-and hkb-MS2 dynamics when light is applied at different developmental times
Sequential movies show tll-MS2 bursts in NC14 and then hkb-MS2 bursts in NC14 when high light is applied starting in either NC13 (top) or NC14 (bottom). Teal and magenta nuclear masks show nuclei with a burst in corresponding frames. Timer (mm:ss) shows time since the start of NC14. Scale bar is 10 µm.

Movie S5: NC14 byn-MS2 dynamics under high in illuminated and unilluminated regions
Movie shows byn-MS2 bursts during NC14 when high light is applied to the illuminated region indicated by the blue bar. The dashed line indicates the illumination boundary. Orange nuclear masks show nuclei with a burst in corresponding frames. Timer (mm:ss) shows time since the start of NC14. Scale bar is 10 µm.